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The present study aimed at the aetiological diagnosis of skin tumours of roe deer (Capreolus capreolus) in the Czech Republic. A total of 33 roe deer specimens showing skin masses were sampled for histopathology, virus detection and identification using polymerase chain reaction (PCR) and deoxyribonucleic acid sequence (DNA) analysis, and to investigate fibropapilloma-associated levels of metallothionein, zinc and oxidative stress in 2012 and 2013. Ticks (Ixodes ricinus) and deer keds (Lipoptena cervi) were also collected and pooled from sampled game specimens. Ticks found on dogs in hunting grounds under study were pooled into another sample. Skin tumours, ranging from 2 to 10 per inspected specimen and 2 to 5 cm in size, were classified as fibropapillomas by histopathology. All 33 viral-infection-suspected skin samples from roe deer in South Moravia and South Bohemia were PCR positive. The nucleotide sequences of PCR products were 100% homologous to the Western roe deer papillomavirus 1 isolate CcPV-1. Ticks and deer keds from positive roe deer and ticks from dogs were PCR and DNA sequence positive for the roe deer papillomavirus. Viral DNA was also demonstrated in one blood sample from a roe deer female. Differences in metallothionein, zinc, taurine and electrochemical index among samples from lesions and normal skin of affected roe deer and negative controls from papillomavirus-non-infected animals were non-significant. While we have demonstrated circulation of specific roe deer papillomavirus in the Czech Republic that results in multiple fibropapillomatous skin tumours, many ecological and epidemiological issues of this wildlife disease still remain unanswered.